Relationships between Plasmodium falciparum incidence and entomologic inoculation rates (EIRs) were determined for a 21-month period in Saradidi, western Kenya, in preparation for malaria vaccine field trials. Children, ranging in age from six months to six years and treated to clear malaria parasites, were monitored daily for up to 12 weeks to detect new malaria infections. Overall, new P. falciparum infections were detected in 77% of 809 children. The percentage of children that developed infections per two-week period averaged 34.7%, ranging from 7.3% to 90.9%. Transmission by vector populations was detected in 86.4% (38 of 44) of the two-week periods, with daily EIRs averaging 0.75 infective bites per person. Periods of intense transmission during April to August, and from November to January, coincided with seasonal rains. Relationships between daily malaria attack rates and EIRs indicated that an average of only 7.5% (1 in 13) of the sporozoite inoculations produced new infections in children. Regression analysis demonstrated that EIRs accounted for 74% of the variation in attack rates. One of the components of the EIR, the human-biting rate, alone accounted for 68% of the variation in attack rates. Thus, measurements of either the EIR or the human-biting rate can be used to predict corresponding attack rates in children. These baseline epidemiologic studies indicate that the intense transmission patterns of P. falciparum in Saradidi will provide excellent conditions for evaluating malaria vaccine efficacy.

Antibodies against repetitive epitopes on Plasmodium falciparum and P. vivax circumsporozoite (CS) proteins and epitopes on the 45-kD and 185–200-kD P. falciparum merozoite surface antigens were measured by radioimmunoassay in Weheragala, a malaria-endemic site in the dry zone of Sri Lanka. Antibodies were measured in sera collected in February at the end of the main malaria transmission season and three months later in May during the low transmission period. Ninety-seven percent of the sample population had antibodies to the P. falciparum CS repeat in February and a significant proportion possessed antibodies directed against all epitopes tested. Concentrations and prevalence of antibodies to the CS repeats decreased with time after the end of malaria transmission in adults and children. Similar temporal changes were observed with antibodies to the epitopes on merozoite surface antigens. Children 7–15 years of age had lower antibody concentrations against most epitopes than adults. Antibody concentrations to two different epitopes within the same merozoite surface antigen showed significant association as did antibody levels against the P. falciparum CS repeat and the predominant P. vivax CS repeat. However, antibody concentrations did not correlate with the presence of blood-stage malaria infections.

Recently, the trend in the number of people traveling from the tropics to malaria-free areas has tremendously increased and this is paralleled by the number of imported malaria cases. Imported infected mosquitoes transmitting the infection to persons living or working nearby international airports have been reported. The possibility that mosquitoes may also reach areas far away from the landing area in the baggage of international travelers is indicated by two cases that are reported here. Malaria should be investigated in any case of unexplained fever even if no apparent risk factor for imported malaria is present, regardless of the distance from international airports.

Although a significant resurgence of malaria in Israel is unlikely at present, the risk for a localized outbreak of malaria cases due to infection of local anopheline mosquitoes by imported cases does exist. A national computerized surveillance system of breeding sites of Anopheles mosquitoes and imported malaria cases was established in 1992 using a geographic information system (GIS). Distances between population centers and breeding sites were calculated, and maps associating epidemiologic and entomologic data were generated. Risk of malaria transmission was assessed with consideration of vectorial capacity and flight range of each Anopheles species. The GIS-based surveillance system ensures that if a localized outbreak does occur, it will be associated rapidly with a likely breeding site, a specific Anopheles vector, and a probable human source, so that prompt control measures can be most efficiently targeted. This cost-effective, GIS-based surveillance system can be expanded and adapted for countries with indigenous malaria transmission.

The role of Didelphis marsupialis as a reservoir of zoonotic hemoflagellates was examined in two ecologically distinct settings in Colombia. While 72% (12 of 18) of the opossums collected in the tropical rain forest harbored Trypanosoma cruzi, other mammals in the area had lower infection rates: 1.3% (Proechymis semispinosus [spiny rat]; 13% Tylomys mirae [climbing rat]; and 6% Rattus rattus). Trypanosoma cruzi isolates from D. marsupialis were similar to zymodeme 1 (Z1), and two of four phenotypes were shared with Tylomys mirae, which is also predominantly arboreal. Terrestrial (P. semispinosus) and peridomestic (R. rattus) animals were infected with Z3 or other Z1 phenotypes, respectively. Schizodeme analysis showed polymorphisms among isolates from mammals, reflecting diverse modes of transmission, and a complex epidemiologic situation. Despite the lower infection rate of the opossum (14%) found in our study in the tropical dry forest as compared with the tropical wet forest, Chagas' disease has been reported only in the former area. This suggests that the lack of alternative blood sources for triatomines of the tropical dry forest, where mammals are less abundant than in the wet forest, may increase the risk of human infection. Among several species of mammals captured in the tropical dry forest, Leishmania chagasi was isolated from 22.7% (5 of 22) D. marsupialis. This finding confirms the important role of opposums in Colombian foci of visceral leishmaniasis, including those where the phlebotomine species involved in transmission is Lutzomyia evansi, an alternative vector to the more common Lutzomyia longipalpis.

Epidemic cholera continues in Peru. Since 1991, cholera surveillance in Peru has been based mainly on clinical recognition. To determine the proportion of reported cholera patients who actually have cholera and to evaluate the clinical case definition used in surveillance, we cultured rectal swabs from patients presenting with acute diarrhea in March 1992 in Trujillo, Peru. Of 197 patients meeting the clinical case definition, 174 (88%) had confirmed Vibrio cholerae O1 infection. In this epidemic setting, watery diarrhea of sudden onset in a person of any age presenting for treatment is highly predictive of cholera. Of note, 90% of the current V. cholerae O1 El Tor isolates were of serotype Ogawa, while a year earlier, all were of serotype Inaba.

For the first time in West Africa, arboviruses were isolated from phlebotomine sand fly pools. One strain of Chandipura virus (a Vesiculovirus), four strains of Saboya virus (a Flavivirus), and one strain of a not yet identified virus were isolated. Three hundred twenty-two pools were established from a population of 33,917 sand flies caught in CO2 light traps in the Ferlo Sahelian region of Senegal from November 1991 to December 1992. This is the first isolation of Chandipura virus from any arthropod in Africa. Saboya virus has already been isolated from small rodents in Senegal; thus, its transmission cycle probably involves rodentophilic sand flies. No strain of Rift Valley fever phlebovirus, which caused an epizootic in this region in 1987, was isolated. During the same time at the same site, 11 sand fly species were identified from 4,191 specimens caught on sticky traps, including Phlebotomus duboscqi, a leishmaniasis vector.

A study of morbidity due to Schistosoma mansoni infection was carried out in Ndombo, a recently established but intense focus in northern Senegal. A random population sample (n = 422) was examined by repeated egg counts, standardized interviews, and clinical examinations. Egg counts were positive in 91%, with more than 1,000 eggs per gram of feces in 41% of the subjects. Abdominal discomfort was reported by 60% of the subjects, diarrhea by 33%; 17% of the stools were liquid upon inspection. Hepatomegaly was mostly mild and found in 7% of the subjects, mainly in males less than 20 years of age. Splenomegaly was detected in only 0.5% of the people examined. There was no significant correlation between the frequency of complaints or symptoms and egg counts. The remarkably mild morbidity in spite of the intense level of many infections may be explained by the recent nature of the focus; more severe chronic morbidity may develop in the future.

Ten Spanish male tourists developed hematospermia and ultrasonographic evidence of involvement of the prostate and/or seminal vesicles after recreational exposure in bodies of fresh water in the Dogon country of Mali. Schistosoma eggs were detected in the ejaculate of five men, in the others, eggs were observed in the urine or feces. Three different species were observed: S. intercalatum, S. haematobium, and S. mansoni. Hemospermia and clinical prostatitis may be frequently unrecognized clinical manifestations of the early stages of infection in previously nonexposed persons. Travelers to endemic areas should be advised on the potential dangers of swimming and other exposure in bodies of freshwater.

Two enzyme-linked immunosorbent assays (ELISA-5B1 using monoclonal antibody [MAb] 114-5B1-A [IgG1] and ELISA-4D12 using MAb 114-4D12-A [IgG3]) that detect circulating soluble egg antigen (CSEA) of Schistosoma mansoni were combined into one assay. This assay showed better performance than either of the two MAbs alone in detecting egg antigen, which was demonstrated with 80 urine samples from patients infected with Schistosoma mansoni from Zaire. The lower detection limit of the combined ELISA was 90 pg of the trichloroacetic acid—soluble fraction of soluble egg antigen (SEA-TCA) per milliliter. Thirty-two serum samples and 107 urine samples from uninfected Dutch individuals were negative when tested with the combined ELISA. This assay showed the same sensitivity (86.3%) with patients' urine samples as parallel testing with ELISA-5B1 and ELISA-4D12 (85%), while ELISA-5B1 and ELISA-4D12 showed sensitivities of 81.3% and 75%, respectively. The sensitivity of the combined ELISA with 51 serum samples was 84.3%, and three of five serum samples available from the seven patients with negative urine were positive for CSEA. The concentration of CSEA calculated from a four-parameters logistic curve for samples tested showed a correlation with egg output and serum circulating anodic antigen (P < 0.0001). Circulating soluble egg antigen in urine showed a significant decrease with an increase in age of the patients in relation to serum CSEA and egg output.

Current diagnosis of Entamoeba histolytica infection requires the direct microscopic identification of the parasite, a technique that is insensitive and cannot distinguish pathogenic E. histolytica from noninvasive E. dispar. Enzyme-linked immunosorbent assay (ELISA) antigen detection tests were developed to distinguish E. histolytica from E. dispar infection in stool specimens. The ELISA result for E. histolytica antigen was positive in 26 of 27 E. histolytica-positive stool specimens, three of 25 E. dispar-positive stools, and one of 30 stools with other or no intestinal parasites, giving a specificity and sensitivity for the detection of E. histolytica infection of 93% and 96%, respectively. The assay result used to detect both E. dispar and E. histolytica was positive in 26 of 27 E. histolytica-positive stools, 19 of 25 E. dispar-positive stools, and one of 30 stools negative by microscopy and culture for Entamoeba, giving a specificity and sensitivity of 97% and 87%, respectively. Because these ELISAs can be completed in several hours, they offer promise as rapid and sensitive means of detecting amebic infection.

Scattered or diffuse myonecrosis is the histopathologic basis for muscle pain and tenderness due to bites by Vipera russelli pulchella in Sri Lanka. These lesions may even occur without any clinical symptoms. Subclinical lesions may form one end of continuous spectrum, with the other being severe pain and muscle tenderness with rhabdomyolysis and myoglobinuria. Electromyographic abnormalities, when present, are suggestive of a myopathic pattern, rather than inflammatory muscle disease. A subclinical motor neuropathy may also occur. Hence, there is evidence for subclinical envenomation following bites by Russell's viper. Early antivenom therapy does not prevent the histologic, electromyographic, or nerve conduction abnormalities.

Overhydration can contribute to fatal complications of falciparum malaria, even though renal function may be normal. In this context, the role of inappropriate secretion of antidiuretic hormone (ADH) has been controversial. Therefore, we have analyzed ADH serum concentrations together with serum osmolality and sodium levels in serum and urine of 17 consecutively studied patients with severe falciparum malaria. Serum sodium levels were low in 13 of 17 patients upon admission and returned to normal levels during antiparasitic therapy. Urine sodium levels were low in seven of 13 patients before treatment and increased during therapy. Urine sodium concentrations were high, however, in the remaining six patients. Serum osmolality was lower in these six patients than in the other seven hyponatremic patients (P < 0.002). In relation to serum osmolality, ADH levels were inappropriately high in these six patients, which confirms the presence of inappropriate secretion of ADH. Serum creatinine levels were not higher in these six patients than in those without inappropriate secretion of ADH. Inappropriate secretion of ADH seemed to be a major cause of hyponatremia, since other factors that could lead to this condition were not found in these six patients. In conclusion, we have shown, that human falciparum malaria can be associated with inappropriate secretion of ADH.

Salmonella typhi, the etiologic agent of typhoid fever, typically has only a phase-1 flagellar antigen, HI-d (fliC). While most strains of S. typhi have HI-d antigen, 10–20% of Indonesian isolates have been reported to possess HI-j antigen instead. To investigate the presence HI-j strains of S. typhi isolates in Korea, where typhoid fever is still a common infectious problem, we used the polymerase chain reaction (PCR) with a pair of oligonucleotides primers that specifically amplified the flagellin gene of S. typhi. Of 375 isolates of S. typhi tested, only one was shown to possess the HI-j antigen, which was shown by the presence of a 1,269-basepair fragment on agarose gel electrophoresis after the PCR. The isolate with the HI-j antigen was cultured from a Korean-Indonesian man who was already symptomatic in Indonesia and was thought to be an Indonesian strain. Because 375 strains tested in this study were collected from cases with typhoid fever in different regions of Korea during the period from 1986 to 1991, it could be concluded that the mutation rate to j antigen is negligible among S. typhi endemic in Korea.

Conventional epifluorescence microscopy (CEM) and confocal laser scanning microscopy (CLSM) were used to visualize the excretory system and the gut on whole organisms of different life-cycle stages of Schistosoma mansoni. To visualize the gut system, an anti-circulating anodic antigen (CAA) monoclonal antibody (MAb) (120-1B10-A) was used, whereas the excretory system was immunohistochemically stained with an antiflame cell MAb (51-4H8-A) and with a recently described anti-egg MAb (114-5B1-A). The CEM procedure resulted in clear images at low magnification but the signal-to-noise ratio on the higher magnification images was very poor. Using CLSM on the adult worm, the 114-581-A MAb demonstrated a well-defined system of canals that could be morphologically identified as the excretory system. The flame cells terminating the branches of the excretory canals showed a clear immunoreactivity with the 114-5B1-A MAb as well as with the specific flame cell MAb. The gut system could be visualized, using an anti-CAA MAb, as two well-defined bands throughout the length of the parasite. Application of the 114-5B1-A MAb on cercariae revealed a strong fluorescence on the cercarial surface, whereas no immunoreactivity could be detected on internal structures. Whole eggs showed a bright fluorescence of the egg shell, whereas miracidia showed immunoreactivity of the germinal cells located in the center of the organism. The CLSM procedure, especially with the recently introduced fast photon-counting option, provides a superior tool to investigate the three-dimensional localization of different epitopes on immunofluorescently stained whole mounts of multicellular organisms in comparison with CEM. The advent of new visualization techniques to exactly localize epitopes of newly discovered MAbs can be of great significance in the development of new screening tests. The identification of epitopes on the different life-cycle stages of this clinically important trematode is valuable for the improvement of therapeutic and prophylactic strategies.

The transformation of the parasite Trypanosoma cruzi from the blood-borne trypomastigote to the intracellular amastigote constitutes a key clinical feature in the pathophysiology of Chagas' disease. That this transition occurs without change in the integrity of the plasma membrane of the parasite suggests the presence of biochemical structures, i.e., signal transduction systems, that convey information regarding the external milieu of the host so as to facilitate this transformation. In higher eukaryotes, it has been found that a heterotrimeric GTP-binding protein (G-protein), composed of αβγ subunits, constitutes a critical component of this complex. Two closely related groups of G-proteins are substrates for cholera toxin (CT)- (Gs) and pertussis toxin (PT)- (Gil-3 and Go) dependent ADP ribosylation. In concert, they link plasma membrane receptors to adenylate cyclase, resulting in the stimulation or inhibition, respectively, of cAMP generation. In this report, we demonstrate the presence of both groups of G-proteins. Cholera toxin-dependent ADP ribosylation of 42- and 45-kD proteins was demonstrable in amastigotes (AMAST), in the cytosol of epimastigotes (EPI), and weakly in trypomastigotes (TRYP), suggesting the presence of the stimulatory GTP-binding protein, Gs, in T. cruzi. Antisera generated against the αs subunit of the Gs heterotrimeric protein (anti-αs) bound to a 45-kD protein CT substrate in the rank order TRYP ≫ AMAST ≈ EPI cytosol. Immunoprecipitation of CT-32P-ADP-ribosylated membranes with anti-αs resulted in 42- and 45-kD proteins. However, no Gs-mediated activation of adenylate cyclase was demonstrable in reconstitution studies using cyc- lymphoma cells, which lack a functional Gs but possess a β-adrenergic receptor and adenylyl cyclase enzyme. Pertussis toxin -catalyzed ADP ribosylation was demonstrable in 39–40-kD particulate proteins of EPI, less strongly in AMAST, and least in TRYP, consistent with the presence of inhibitory (Gi) and Go GTP-binding proteins. In support of this observation, immunochemical analysis of the PT substrates identified the presence of αo and αi1-2-3 in EPI, AMAST and TRYP, although, with the exception of αi3, both toxin and associated immunochemical PT substrates are decreased in AMAST and TRYP relative to EPI. Although the functions of these putative G-proteins in T. cruzi are still unclear, their expression may be regulated by the state of parasite differentiation. Despite our inability to demonstrate a function for these G-proteins in the adenylate cyclase signal transduction system, their presence in T. cruzi suggests that they may function in other signal transduction pathways yet to be elucidated. Future investigations of their function may reveal important targets for therapeutic intervention in this disease.

Recombinant DNA probes from a genomic Leishmania major library were screened for their potential to distinguish among Old World Leishmania taxa by Southern blot analysis. A probe, pDK10, was selected and tested on a panel of 58 Old World Leishmania strains that had already been typed isoenzymatically; these strains belong to the different species described so far and had been isolated from various hosts and vectors in 14 countries. In the present study, 45 zymodemes were represented. Using the pDK10 probe, we were able to differentiate between the different phenetic complexes. No variations in hybridization patterns were found within these complexes. In addition, there was a good concordance between identification based on DNA hybridization with the pDK10 probe and that based on isoenzyme typing. The probe has been applied in identifying Leishmania strains that were isolated in Tunisia from humans, animals, or insects. Our results show that the application of the pDK10 probe, in combination with a Pst I digestion of Leishmania DNA, could be a possible alternative to isoenzyme analysis for the identification of Leishmania strains.

New drugs for causal prophylaxis of malaria are needed. A proguanil/sulfamethoxazole combination was investigated using a rhesus monkey model (Macaca mulatta infected with Plasmodium cynomolgi) to determine whether causal prophylaxis could be achieved. When a five-day regimen of proguanil (40 mg/kg/day) combined with sulfamethoxazole (100 mg/kg/day) was used, infection of all animals (6 of 6) was observed, with an extended prepatent period (median 40 days). Two control animals became infected on days 9 and 23 following sporozoite inoculation. Plasma concentrations indicated that proguanil and sulfamethoxazole were adequately absorbed and metabolized to cycloguanil and N4-acetylsulfamethoxazole, respectively. Analysis of liver biopsy specimens demonstrated that the drugs were present two days following sporozoite inoculation but were not detectable one week later. Proguanil plus sulfamethoxazole does not eliminate exoerythrocytic-stage parasites in the rhesus monkey-P. cynomolgi model.

The sporontocidal activity of three 8-aminoquinolines, a 1,4-naphthoquinone, and three dihydroacridine-diones was determined against the ANKA clone of Plasmodium berghei and both chloroquine-sensitive (NF54) and chloroquine-resistant (7G8) P. falciparum, Anopheles stephensi mosquitoes previously fed on P. berghei-infected mice or P. falciparum-infected cultures were refed on uninfected mice treated previously with a given drug. Sporontocidal activity was determined by assessing both oocyst and sporozoite development. Neither primaquine nor menoctone exhibited sporontocidal activity against P. berghei or either strain of P. falciparum at a dose of 100 mg base drug/kg mouse body weight, whereas the other five compounds each effectively interrupted the sporogonic development of all three parasite strains at this dose. These data clearly demonstrate that experimental dihydroacridine-diones and 8-aminoquinolines are capable of interrupting the sporogonic development of P. berghei and chloroquine-sensitive and chloroquine-resistant P. falciparum. These data also suggest that the P. berghei model may be used to accurately predict sporontocidal activity against P. falciparum.